Vegetable oils are utilized not only in the food industry, but also increasingly in the chemical industry. The utility of any particular oil depends upon chemical and physico-chemical properties of the oil, which is determined by the composition of the constituent fatty acids. Plant oils are often modified to meet industrial specifications. Such modification of vegetable oil has typically been achieved by chemical means (fractionation, interesterification, hydrogenation, or other chemical derivatization), but genetic means (plant breeding, mutagenesis and genetic engineering) are increasingly being used to provide novel oil feedstocks.
One class of particular interest is the class of fatty acids containing three carbon carbocyclic rings, which includes the cyclopropane fatty acids (CPA-FAs) and cyclopropene fatty acids (CPE-FAs). The cyclopropene ring confers two unique properties for these fatty acids and oils. First, hydrogenation produces large amounts of methyl-branched fatty acids. These will give the low temperature properties equivalent to unsaturated fatty acids and their esters without the oxidative susceptibility of the double bonds, and therefore may find uses in lubrication and related fields (Kai, Y. (1982) J. Am. Oil Chem. Soc. 59: 300-305). Moreover, the methyl-branched fatty acids may also be viewed as a replacement for isostearic acids formed in dimer acid production, and isostearic is an article of commerce in the oleochemical industry where it is used in applications as diverse as cosmetics and lubricant additives. Second, the cyclopropene ring is highly strained and readily ring opens in an exothermic reaction with electrophiles. Oils with high levels of cyclopropene fatty acid, such as Sterculia foetida oil, self-polymerize at elevated temperatures. This Property is Particularly Applicable to the Production of Coatings and polymers. In the Sterculia foetida oil, sterculic acid reacts with acetic acid to produce a variety of acetyl esters, as well as with short or medium chain saturated fatty acids to yield monounsaturated estolide products (Kircher (1964) J. Org. Chem. 29:1979-1982); all of these products can be further hydrogenated and saponified or hydrolyzed to form hydroxy fatty acids. Reaction of the oil with dibasic carboxylic acids should result in polymers. Moreover, sterculic acid might also be used as a biocide in fatty acid soap formulation.
On the other hand, CPE-FAs are considered an anti-nutritional factor in food oils. Many seed lipids containing CPE-FAs are extensively consumed by humans, especially in tropical areas (Ralaimanarivo et al. (1982) Lipids 17 (1): 1-10). It is well documented that dietary CPE-FAs lead to the accumulation of hard fats and other physiological disorders in animals (Phelps et al. (1965) Poultry Science 44: 358-394; Page et al. (1997) Comparative Biochemistry And Physiology B-Biochemistry & Molecular Biology 118 (1): 79-84). CPE-FAs are strong inhibitors of variety of desaturases in animals (Cao et al (1993) Biochimica et Biophysica Acta 1210 (1): 27-34; Fabrias et al. (1996) Journal of Lipid Research 37 (7): 1503-1509; Fogerty et al. (1972) Lipids 7(5): 335-338), which might be the cause of at least some of the observed disorders. Because of these health concerns, vegetable oils containing CPE-FAs must be treated with high temperature or hydrogenation before consumption. These treatments add to the oil processing costs, and also result in the presence of a certain percentage of trans fatty acids produced due the hydrogenation; the presence of such trans fatty acids are also undesirable. Therefore, it would be desirable to obtain plant oils with greatly reduced levels of CPE-FAs, as the availability of such oils would significantly reduce the processing costs, decrease the presence of undesirable hydrogenated fatty acids, and enhance the value of the oils for food consumption. Elimination of CPE-FAs would also enhance the value of unprocessed seeds or seed meal, such as cottonseed, as animal feed.
Currently, there are no commercial sources of oils rich in CPE-FAs. It is believed that plant CPE-FAs are synthesized from CPA-FAs via desaturation. E. coli and other bacteria have the ability to synthesize fatty acids containing a cyclopropane ring. The reaction is catalyzed by the enzyme cyclopropane fatty acid synthase (also known as cyclopropane synthase or unsaturated phospholipid methyltransferase; E.C. 2.1.1.16) and involves the addition of a methylene group from S-adenosylmethionine across the double bond of phospholipid hexadecenoyl or octadecenoyl groups. CPA-FAs (CFAs), such as dihydrosterculate (DHS) are characterized by a saturated 3-membered ring, as shown by the following structure, where X═OH for a free fatty acid, or an alcohol moiety for an ester:
The cyclopropane fatty acid synthase gene in E. coli has been cloned and sequenced (Grogan et al. (1997) J. Bacteriol. 158:286-295 and Wang et al. (1992) Biochemistry 31: 11020-11028). No CPE-FAs have been reported in bacteria.
CPA-FAs (CPA-FA) and CPE-FAs (CPE-FA) are not widely distributed in high plants, but they are found in the seed oils of limited families, including the Malvaceae, Sterculiaceae, Bombaceae, Tilaceae, Mimosaceae and Sapindaceae (Smith (1970) Progress in the Chemistry of Fats and Other Lipids (Pergamon Press: New York) Vol. 11, pp 139-177; Christie (1970) in Topics in Lipid Chemistry (Gunstone F D Ed.; Logos Press: London) Vol. 1, pp 1-49; Badami and Patil (1981) Prog. Lipid Res. 19: 119-153). The CPA-FAs and CPE-FAs are not confined to seeds. Kuiper and Stuiver ((1972) Plant Physiol. 49: 307-309) have described long-chain CPA-FAs in various polar lipid classes of leaves of early spring plants. Yano et al. ((1972) Lipids 7: 30-34) and Schmid and Patterson ((1988) Phytochem. 27: 2831-2834) report that CPA-FAs and CPE-FAs are found in root, leaf stem and callus tissue in plants of the Malvaceae.
In a few plant species, CPA-FAs can reach high levels, in other words up to 40% in Litchi chinensis (Vickery et al. (1980) J. Am. Oil Chem. Soc. 57: 87-91; and Gaydou et al. (1993) J. Ag. Food Chem. 41: 886-890). However, it is more common to find CPE-FAs, particularly in the order Malvales (for example, as in the report by Bohannon and Kleiman (1978) Lipids 13: 270-273), and a biosynthetic pathway of CPE-FAs through CPA-FAs was postulated by Yano et al. ((1972) Lipids 7: 35-45). Thus, in plants, CPE-FAs exist primarily in the form of sterculic and malvalic acids, where malvalic acid is the one carbon homolog of sterculic acid and is obtained by chain shortening at the carboxyl end by ∀-oxidation. The CPE-FAs are usually accompanied with small amount of corresponding CPA-FAs, dihydrosterculic and dihydromalvalic acids. However, there have been no confirmed identified and isolated plant genes which encode proteins which are capable of synthesizing CPA-FAs. Moreover, plants with high levels of cyclopropene are not grown commercially.
Therefore, it would be desirable to be able to generate vegetable oils with high amounts of cyclopropane and CPE-FAs. One route is by identifying and isolating a plant gene which is capable of synthesizing CPA-FAs. Such a gene could then be used to transform oil crop plants. Identification of such a gene could also be used to reduce the levels of CPE-FAs by gene silencing techniques.